This invention relates to a method and apparatus for capillary electrophoresis with fraction collection onto a porous substrate. More particularly, this invention relates to an improved method and apparatus for capillary electrophoresis that permits isolation of solutes from a solution onto a porous substrate after separating the solutes by capillary electrophoresis.
Capillary electrophoresis (CE) is an efficient analytical separation technique for analysis of minute amounts of sample. CE separations are performed in a narrow diameter capillary tube, which is filled with an electrically conductive medium termed the "carrier electrolyte." An electric field is applied between the two ends of the capillary tube, and species in the sample move from one electrode toward the other electrode at a rate which is dependent on the electrophoretic mobility of each species as well as on the rate of bulk fluid movement in the tube. CE may be performed using gels or liquids in the capillary. In one liquid mode, known as free zone electrophoresis separations are based on differences in the free solution mobility of sample species. In another liquid mode, micelles are used to effect separations based on differences in hydrophobicity. This is known as Micellar Electrokinetic Capillary Chromatography. In capillary gel electrophoresi the capillary tube is filled with an electrically conductive gel rather than with liquid electrolyte. The gel functions as an anticonvective support to minimize sample band spreading. In free zone capillary electrophoresis, a high molecular weight solute such as a polyethylene oxide or hydroxymethyl cellulose can be added to the solvent to provide seiving effects analagous to that supplied by a gel.
CE is advantageous for several reasons. These include fast separation speed, high resolution and small sample size. For example, separation speeds using CE can be 10 to 20 times faster than conventional gel electrophoresis. In part, high resolution can be obtained through the use of high voltages because of the rapid dissipation of heat by the capillary. Further, band broadening due to convection is minimized due to the narrow capillary inner diameter. In most forms of CE, the lack of an anticonvective gel medium eliminates variable sample path tortuosity as a band spreading cause. In electrophoresis, the phenomenon of electroosmosis, or electroosmotic flow (EOF) occurs. This is a bulk flow of liquid which can move in either direction within the capillary tube and which affects all of the sample molecules regardless of charge. EOF ca contribute to improved resolution or separation speed in free-zone CE.
At the present time, fraction collection for CE is generally effected by inserting the exit end of the capillary tube into a vial containing electrolyte solution. An electrode is also inserted into the vial and the separation voltage is activated to effect migration of the solute from the capillary into the vial. After a period of time, the capillary tube end is moved to another vial and the process is repeated. This process is undesirable since the sample becomes diluted by the electrolyte in the vial. In addition, it is difficult to determine when to transfer the capillary exit end to another vial since, in general, one is not able to monitor when a sample portion has completely exited the capillary tube, particularly when the sample bands are closely spaced. One approach for overcoming this problem was reported by Huang and Zare at the High Performance Capillary Electrophoresis Symposium in January, 1990. The capillary is modified adjacent to the exit end by forming a hole in the capillary wall, making a porous frit in the hole and surrounding the porous frit with a reservoir filled with electrolyte. Electrical contact is made to the capillary through the fritted hole by means of an electrode in the reservoir filled with electrolyte. The exit en of the capillary is contacted with a rotating drum covered with filter paper. Most of the liquid in the Capillary is carried past the porous frit by electroosmotic flow onto the filter paper. This approach is undesirable since it is difficult and time consuming to construct the fritted hole. Also, a portion of the sample is lost from the capillary through the frit and into the reservoir. Also, this technique only function when an electrosmotic flow occurs toward the capillary exit.
U.S. Pat. Nos. 4,631,120 and 4,631,122 disclose a method and apparatus for effecting electrophoresis through a porous substance such as a gel or paper. The porous substance rather than a liquid is required as an anticonvective medium. In one mode, the porous substance is positioned vertically and wherein one liquid electrolyte reservoir is positioned above the porous substance and a second liquid electrolyte reservoir is positioned below the porous substance. In a second configuration the porous substance is positioned horizontally in a open slot. This method does not permit the use of a liquid transport medium since the medium would become admixed with the electrolytes within which the electrodes are immersed. The disadvantages of this method and apparatus are the same as that encountered in gel electrophoresis not utilizing a capillary, namely; poor rejection of heat generated during the electrophoresis process, slow rate of separation spreading, band spreading caused by differential tortuous paths for the sample within the gel and the requirement of manual sample loading. In addition, in both cases the end of the transport medium is immersed in an electrolyte and a portion of the sample migrating to the collection membrane can be lost in the electrolyte. In addition, the detection mean is off line and usually is nonquantitative.
U.S. Pat. No. 4,735,697 disclose a method and apparatus for separating complex mixtures of biomolecules such as proteins or nucleic acids in two stages. In the first stage, the mixture is separated by high performance liquid chromatography (HPLC). The effluent from the HPLC stage is treated so that the components exhibit a uniform surface charge such as by contact with a detergent. The treated HPLC effluent then is deposited on the surface of an electrically charged separation gel. This system does not permit high resolution spatial collection of sample since the electric field within the separation gel is not focussed at the HPLC column exit.
Western blotting is a technique by which samples separated in a gel during a prior electrophoresis step are transferred to a membrane. The membrane and gel are positioned between two plate electrodes and the membrane is wet with an electrolyte such as with a wet absorbent layer positioned in contact with the membrane. The samples in the gel are caused to migrate to the membrane by the voltage applied between the plate electrodes.
It would be desirable to provide a means for recovering separated samples from a capillary electrophoresis process which does not dilute the sample and which avoids sample loss. In addition it would be desirable to provide such a means which is simple to produce and operate. It would also be desirable to provide a means for collecting a sample in a format which is compatible with present analysis techniques, such as amino acid sequencing, radio immuno assay, ELISA or the like. It would also be desirable to provide a means that preserves the spatial resolution of the CE process in the recovered separated sample species.